1. Field of the Invention
Medical diagnosis is dependent to a great degree on the determination of the presence or absence of various compounds or their amounts in physiological fluids. Forensic medicine is also frequently concerned with the determination of the presence or absence of various physiologically active materials. Many systems have been developed to be able to accurately determine the presence and amounts of materials of interest.
One particular group of techniques is referred to as immunoassays. The immunoassays are dependent upon the recognition capability of a molecule, which may be referred to as a receptor, and is normally an antibody. Vertebrates have the capacity to produce molecules of protein which are able to distinguish a compound or group of compounds from other compounds which have similar or different structures. One immunoassay method, which has been available for a long period of time, is radioimmunoassay. Radioimmunoassay employs a molecule which has been tagged with a radioactive isotope. By combining an antibody to the ligand (ligand is the compound to be determined) with a small amount of ligand which has been tagged with a radioactive isotope and separating ligand bound to antibody from ligand which is unbound, the amount of radioactive ligand remaining in the supernatant solution will be related to the amount of ligand which was present in the original sample. Since this technique requires a separation into two phases, it will be referred to as a heterogeneous immunoassay technique.
Recently, two homogeneous immunoassay techniques have been developed by Syva Company, sold under the trademarks, FRAT, and EMIT. By homogeneous it is intended that after mixing the reagent, the assay does not require a separation step. The first homogeneous assay, the FRAT technique, uses a stable free radical tag, rather than a radioactive tag on the ligand. The ESR spectrum changes significantly in relation to the rate of tumbling of the free radical in solution. When the labeled ligand is bound to antibody, the peak height is substantially lower than when the labeled ligand freely tumbles in solution. Since the amount of labeled ligand bound to antibody will be related to the amount of ligand in solution, the peak height obtained with the assay solution can be related to known standards, and the concentration of the ligand determined.
The second homogeneous immunoassay involves an enzyme tag. The reagent employed has ligand bound to enzyme at a position, so that when antibody is bound to the ligand, the activity of the enzyme is substantially reduced. Thus, one can assay for enzyme activity in the assay solution, with the enzyme activity being related to the amount of ligand which was present in the unknown.
All of these techniques have been found to be highly sensitive, being able to detect concentrations of ligand at 10.sup..sup.-6 M or below, and applicable to a wide variety of haptens and antigens. However, as is evident from the above discussion, a different detector molecule must be prepared for each ligand to be determined. While for many ligands no significant difficulty is encountered, with some ligands, substantial difficulty is encountered in preparing the detector molecule. It would be of great convenience if a single detector molecule could be prepared, which had the desired properties and could be used for detecting a wide range of ligands.